Purification, crystallization and preliminary X-ray investigation of the complex of human vitamin D binding protein and rabbit muscle actin.

The vitamin D binding protein binds globular actin with high affinity and is involved in the clearance of actin from the blood circulation. A complex of the human vitamin D binding protein and rabbit muscle actin was subjected to purification steps. The pure complex was crystallized using the hanging-drop vapour-diffusion procedure. The best obtained crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 74.44, b = 74.90, c = 88.02 A, beta = 110.19 degrees. A complete data set to 2.4 A was collected from a single crystal using synchrotron radiation at DESY, Hamburg, Germany.

Relationship between free and total 1,25-dihydroxyvitamin D in conditions of modified binding.

OBJECTIVE: A novel assay was employed to study the free 1,25-dihydroxyvitamin D (1,25(OH)2D) concentrations in various populations with different levels of 1,25(OH)2D and vitamin D binding protein (DBP). DESIGN: In 12 healthy women before and after 3 months of oral estrogen/progestagen treatment, 10 pregnant women, and 16 patients with a nephrotic syndrome and normal renal function, the concentrations of total and free 1,25(OH)2D, DBP and albumin were assessed. METHODS: The total concentration of 1,25(OH)2D in serum was assessed using a radioreceptor assay. The free fraction of 1,25(OH)2D was measured using symmetric dialysis. DBP was assessed using single radial immunodiffusion. Serum albumin concentrations were measured on an automated analyzer. RESULTS: In healthy women, the concentrations of total 1,25(OH)2D, free 1,25(OH)2D and DBP were 132+/-19 pmol/l, 92+/-30 fmol/l and 5.59+/-0.43 micromol/l. After 3 months of estrogen/progestagen treatment, total 1,25(OH)2D and DBP levels rose significantly to 175+/-51 pmol/l and 8.32+/-1.59 micromol/l (P< or =0.05 and P< or =0.001); the free 1,25(OH)2D remained unchanged (105+/-39 fmol/l; not significant). Pregnant women had significantly higher levels of total 1,25(OH)2D and DBP (239+/-68 pmol/l and 11.32+/-1.77 micromol/l; both P

Biological activity of CD-ring modified 1alpha,25-dihydroxyvitamin D analogues: C-ring and five-membered D-ring analogues.

Nonsteroidal analogues of 1alpha,25(OH)2D3, lacking either the full five-membered D ring (C-ring analogues) or the full six-membered C ring (D-ring analogues) are more potent inhibitors of cell proliferation or inducers of cell differentiation than is 1alpha,25(OH)2D3. Maximal superagonistic activity was seen for the C-ring analogue with a 24(R)-hydroxyl group in the side chain [30- to 60-fold the activity of 1alpha,25(OH)2D3]. The 19-nor-16-ene-26,27-bishomo C-ring analogue showed the best ratio of antiproliferative to calcemic effects (1275-fold better than 1alpha,25(OH)2D3 and severalfold better than all vitamin D analogues so far described). The analogues are able to stimulate specific vitamin D-dependent genes and are active in transfection assays using an osteocalcin promoter VDRE. Low binding affinity to the vitamin D binding protein, differences in metabolism, or affinity for the vitamin D receptor (VDR) are not the most important explanations for the enhanced intrinsic activity. However, the analogues are able to induce conformational changes in the VDR, which makes the VDR-ligand complex more resistant against protease digestion than is 1alpha,25(OH)2D3. In contrast to 20-epimer steroidal vitamin D analogues, 20-epimer C-ring analogues were less potent than analogues with a natural C-20 configuration. In conclusion, several nonsteroidal vitamin D analogues are superagonists of 1alpha,25(OH)2D3 despite lower receptor affinity and, for the C-ring analogues, higher flexibility of the side chain; moreover, they have a better selectivity profile than all analogues yet published.

The biological activity of nonsteroidal vitamin D hormone analogs lacking both the C- and D-rings.

1alpha,25-dihydroxyvitamin D is a key calcium-regulating hormone but also displays potent differentiating and antiproliferative activities on many cell types. The structural requirements of this secosteroid hormone have been extensively studied for the A-ring and side chain, whereas relatively little is known about the requirements of the natural CD-ring structure for the vitamin D-like biological activity. We have embarked on a vast program in which derivatives were synthesized and evaluated characterized by profound structural changes in the central C/D-region. This first series of nonsteroidal analogs consists of (1R,3S)-5-((Z,2E)-4-((1S,3S)-3-(4-hydroxy-4-methylpentyl)-1,2,2-++ +trimethylcyclopentyl)-2-butenylidene)-4-methylenecyclohexan e-1,3-diol (KS 176) and derivatives thereof. These analogs are characterized by the absence of normal C- and D-rings and by the presence of an unnatural five-membered ring which we call the E-ring. KS 176 with the otherwise natural side chain structure of 1alpha,25(OH)2D3 has between 10 and 30% of the biological activity of 1alpha,25(OH)2D3 when tested in vitro (prodifferentiating effects on HL-60 and MG-63; antiproliferating activity on MCF-7 and keratinocytes) but has minimal in vivo calcemic effects. Introduction of several side chain modifications created analogs with increased intrinsic noncalcemic biological properties, whereas their calcemic potency remains very low. These data demonstrate that the full CD-rings are not mandatory for the biological activity of 1alpha,25(OH)2D3 since they can be replaced by a new ring structure which generates an appropriate spacing of the A-seco B-rings in relation to the side chain. The biological activity of these nonsteroidal analogs probably involves a classical genomic activation since they are also active in transfection assays using an osteocalcin vitamin D responsive element coupled to a human growth hormone reporter gene.

Comparison of 6-s-cis- and 6-s-trans-locked analogs of 1alpha,25-dihydroxyvitamin D3 indicates that the 6-s-cis conformation is preferred for rapid nongenomic biological responses and that neither 6-s-cis- nor 6-s-trans-locked analogs are preferred for genomic biological responses.

The hormone 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] generates biological responses via both genomic and rapid, nongenomic mechanisms. The genomic responses utilize signal transduction pathways linked to a nuclear receptor (VDRnuc) for 1alpha,25(OH)2D3, while the rapid responses are believed to utilize other signal transduction pathways that may be linked to a putative membrane receptor for 1alpha,25(OH)2D3. The natural seco steroid is capable of facile rotation about its 6,7 single carbon bond, which permits generation of a continuum of potential ligand shapes extending from the 6-s-cis (steroid like) to the 6-s-trans (extended). To identify the shape of conformer(s) that can serve as agonists for the genomic and rapid biological responses, we measured multiple known agonist activities of two families of chemically synthesized analogs that were either locked in the 6-s-cis (6C) or 6-s-trans (6T) conformation. We found that 6T locked analogs were inactive or significantly less active than 1alpha,25(OH)2D3 in both rapid responses (transcaltachia in perfused chick intestine, 45Ca2+ influx in ROS 17/2.8 cells) and genomic (osteocalcin induction in MG-63 cells, differentiation of HL-60 cells, growth arrest of MCF-7 cells, promoter transfection in COS-7 cells) assays. In genomic assays, 6C locked analogs bound poorly to the VDRnuc and were significantly less effective than 1alpha,25(OH)2D3 in the same series of assays designed to measure genomic responses. In contrast, the 6C locked analogs were potent agonists of both rapid response pathways and had activities equivalent to the conformationally flexibile 1alpha,25(OH)2D3; this represents the first demonstration that 6-s-cis locked analogs can function as agonists for vitamin D responses.

Nonhypercalcemic vitamin D analogs: interactions with the vitamin D-binding protein.

The natural vitamin D hormone, 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3), not only regulates serum and bone calcium homeostasis but is probably also a paracrine factor in several cells and tissues including skin, immune system, placenta and brain, where it stimulates cell differentiation and inhibits cell proliferation. Several structural analogs of 1 alpha, 25-(OH)2D3 not only have superagonist activity but also display a selective action profile: indeed they maintain or have increased activity on cell differentiation/proliferation but also have substantially decreased calcemic activity when compared to 1 alpha,25-(OH)2D3. This decreased calcemic activity is partially due to mere pharmacological reasons: because of low binding affinity for the plasma vitamin D-binding protein, a more rapid extracellular metabolism and increased cellular uptake is possible when compared to 1 alpha,25-(OH)2D3. Their short extracellular half-life combined with comparable or enhanced transactivation potency together with analog-and cell-type-specific intracellular metabolism can probably explain why some analogs have a unique combination of superagonist activity and specific action profile with favorable dissociation of differentiation versus calcemic potency.

Crystallization and X-ray investigation of vitamin D-binding protein from human serum. Identification of the crystal content.

Vitamin D-binding protein (DBP), a multifunctional, highly polymorphic glycoprotein responsible for the transport of vitamin D and for sequestering extracellular actin, was isolated from human serum and crystallized using vapour diffusion methods. The crystals were grown from 7.5% v/v polyethylene glycol 400 and 0.1 M acetate buffer at pH 4.6. These crystals show diffraction patterns consistent with the tetragonal space groups P4(1) and P4(3) with unit cell dimensions a = b = 135.5(4) A and c = 75.9(4) A. They diffract to 2.3 A. Using polyacrylamide gel electrophoresis it was shown that according to their electrophoretic mobility the O-glycosylated isoforms, with a terminal sialic acid residue, are absent in the crystals.

Decreased cortisol-binding affinity of transcortin Leuven is associated with an amino acid substitution at residue-93.

Genomic DNA was isolated from two related individuals who are homozygous for transcortin Leuven, a corticosteroid-binding globulin variant with decreased cortisol-binding affinity. This material was amplified using intron-specific oligonucleotide primers in a polymerase chain reaction to obtain the four exons that encode transcortin. Sequence analysis of these exons showed several mutations within the coding sequence of both individuals, but only one of these will result in an amino acid substitution. This mutation is located within exon 2 and alters the codon (CTC) normally associated with Leu-93 in the transcortin polypeptide to a codon (CAC) for histidine in the variant genes.

Molecular analyses of a human sex hormone-binding globulin variant: evidence for an additional carbohydrate chain.

Genomic DNA was isolated from an individual who is homozygous for a sex hormone-binding globulin (SHBG) variant that resolves into three molecular weight forms of 56K, 52K, and 48K during electrophoresis under denaturing conditions (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). This material was amplified using intron-specific oligonucleotide primers in a polymerase chain reaction to obtain the eight exons encoding SHBG. Sequence analysis of these exons revealed a point mutation encoding an amino acid substitution (Asp --> Asn) at residue 327 in the SHBG polypeptide, and the same mutation was identified in three siblings who also appear to be homozygous for this trait. This mutation introduces an additional consensus site for N-glycosylation at this position, and to confirm its utilization we introduced it into a human SHBG complementary DNA. The mutated complementary DNA was inserted into the pRc/CMV expression vector, and transfected into Chinese hamster ovary (CHO) cells. The product was secreted normally but proportionally less of it (54%) bound to concanavalin A when compared to normal SHBG produced by CHO cells (85%), or SHBG in the serum of either a normal individual or those who produce an electrophoretic variant (98%). Furthermore, when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, the SHBG variant produced by CHO cells consisted of a 60K subunit as well as the heavy (52K) and light (48K) subunits associated with normal SHBG produced by CHO cells or in serum. This additional subunit is larger than the variant in serum and probably reflects a greater degree of complexity in the carbohydrate structures added to recombinant SHBG during synthesis in CHO cells. Nevertheless, its steroid-binding affinity was equal to normal SHBG produced by CHO cells or SHBG in serum.

Homologous radioimmunoassay of human osteocalcin.

Osteocalcin or bone gamma-glutamic acid-containing protein (GLA protein) was isolated from human bone and used to develop a homologous radioimmunoassay of human osteocalcin. The effect of age on serum osteocalcin was studied in 380 normal children and adolescents and 330 normal adults. The mean (+/- SD) values in adults were higher in men [25 +/- 5 micrograms/L (4.3 +/- 0.8 nmol/L)] than in premenopausal women [20 +/- 6 micrograms/L (3.4 +/- 1.0 nmol/L); P < 0.01], but both were lower than in postmenopausal women [29 +/- 2 micrograms/L (5.0 nmol/L)]. The highest concentrations were seen in girls [ages 10-12 years: 99 +/- 38 micrograms/L (17.0 nmol/L)] and boys [ages 14-16 years: 107 +/- 57 micrograms/L (18.4 nmol/L)]. These mean values were substantially higher than those previously reported for results of heterologous osteocalcin radioimmunoassays but the correlation (r = 0.87, n = 77, P < 0.001) between both sets of results was excellent. In patients with metabolic bone diseases characterized by high or low bone turnover, the increase or decrease in serum osteocalcin observed was as expected. This homologous radioimmunoassay of human osteocalcin thus reflects bone turnover but reports serum concentrations higher than previously suspected.

Polyunsaturated fatty acids decrease the apparent affinity of vitamin D metabolites for human vitamin D-binding protein.

The affinity of purified human vitamin D-binding protein from serum (DBP) for 25-hydroxyvitamin D3 (25-OHD3) and 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] was measured in the presence of free fatty acids (FFA), cholesterol, prostaglandins and several drugs. Mono- and polyunsaturated fatty acids markedly decreased the affinity of both 25-OHD3 and 1,25-(OH)2D3 for DBP, whereas saturated fatty acids (stearic and arachidic acid), cholesterol, cholesterol esters, retinol, retinoic acid and prostaglandins (A1 and E1) did not affect the apparent affinity. Several chemicals known to decrease the binding of thyroxine to its plasma-binding protein did not affect the affinity of DBP. The apparent affinity of DBP for both 25-OHD3 and 1,25-(OH)2D3 decreased 2.4- to 4.6-fold in the presence of 36 microM of linoleic or arachidonic acid, respectively. Only a molar ratio of FFA:DBP higher than 10,000 was able to decrease the binding of 25-OHD3 to DBP by 20%. Much smaller ratio's of FFA:DBP (25 for arachidonic and 45 for oleic acid), however, decreased the binding of 1,25-(OH)2D3 to DBP. These latter ratio's are well within the physiological range. The addition of human albumin in a physiological albumin:DBP molar ratio did not impair the inhibitory effect of linoleic acid on the binding of [3H]25-OHD3 to DBP. The binding and bioavailability of vitamin D metabolites thus might be altered by mono- and polyunsaturated but not by saturated fatty acids.

Genetic variation of human sex hormone-binding globulin: evidence for a worldwide bi-allelic gene.

Genetic variation of human sex hormone-binding globulin (SHBG) has been investigated on 1690 unrelated neuraminidase-treated serum samples using isoelectric focusing followed by transfer to nitrocellulose membranes and immunostaining. Three clearly distinct isoelectric focusing patterns, consistent with the expression of an autosomal genetic system, were identified. Using allele frequencies, calculated on the basis of a bi-allelic gene, an excellent agreement between observed and expected phenotype numbers was obtained in every examined population sample. Family data along with the observed distribution of the three SHBG phenotypes among racially different groups and sexes indicate that SHBG is worldwide encoded by two autosomal codominant alleles. Compared with healthy Belgian blood donors no statistically significant differences were noted for the allele frequencies among 399 patients and 70 hirsute women of Belgian origin. Evidence is also presented that the subunit produced by the variant allele (SHBG2) has a higher molecular mass than the one produced by the regular allele (SHBG1) and that the three SHBG genotypes have identical binding characteristics for 5 alpha-dihydrotestosterone.

Regulation of calbindin D 28K and its mRNA in the intestine of the domestic hen.

Intestinal calbindin synthesis in laying hens was analyzed to assess controlling factors operating during egg formation. In the absence of vitamin D, calbindin was not induced by estrogen and testosterone. In immature vitamin D-replete pullet, blood levels of 1,25(OH)2D3 increased in response to estrogen but the duodenal concentration of calbindin and its mRNA were increased only when testosterone was given together with estrogen. The plasma concentration of 1,25(OH)2D3 and the duodenal levels of calbindin and its mRNA were substantially higher in laying hens than in immature pullets. No differences in these parameters were observed between the stages of the ovulatory cycle. Suppression of shell formation for a week decreased the concentration of 1,25(OH)2D3 and of duodenal calbindin but did not affect the level of its mRNA. When egg shell formation resumed in hens previously laying shell-less eggs, the concentrations of 1,25(OH)2D3 and of calbindin and its mRNA increased toward the end of shell formation. A most important factor regulating intestinal calbindin synthesis in laying hens turned out to be 1,25(OH)2D3. Intestinal calbindin mRNA is more stable in laying hens than in young birds as its concentration declines more slowly when the stimulation provided by 1,25(OH)2D3 is withdrawn, as occurs following suppression of shell formation and after parathyroidectomy in laying birds. Intestinal calbindin mRNA is therefore increased by a process other than increasing 1,25(OH)2D3 formation. The factor influencing the stability of this mRNA in laying hens could be calcium. It is concluded that in hens the increased duodenal calbindin synthesis elicited by plasma 1,25(OH)2D3 at sexual maturity primarily involves a transcriptional process and the stabilization of the mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure function analysis of vitamin D analogs with C-ring modifications.

Analogs of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3) with substitutions on C-11 were synthesized. Small apolar substitutions (11 alpha-methyl, 11 alpha-fluoromethyl) did not markedly decrease the affinity for the vitamin D receptor, but larger (11 alpha-chloromethyl or 11 alpha- or 11 beta-phenyl) or more polar substitutions (11 alpha-hydroxymethyl, 11 alpha-(2-hydroxyethyl] decreased the affinity to less than 5% of that of 1 alpha,25-OH)2D3. Their affinity for the vitamin D-binding protein, however, increased up to 4-fold. The biological activity of 11 alpha-methyl-1 alpha,25-(OH)2D3 closely resembled that of the natural hormone on normal and leukemic cell proliferation and bone resorption, whereas its in vivo effect on calcium metabolism of the rachitic chick was about 50% of that of 1 alpha,25-(OH)2D3. The 11 beta-methyl analog had a greater than 10-fold lower activity. The differentiating effects of the other C-11 analogs on human promyeloid leukemia cells (HL-60) agreed well with their bone-resorbing activity and receptor affinity, but they demonstrated lower calcemic effects in vivo. Large or polar substitutions on C-11 of 1 alpha,25-(OH)2D3 thus impair the binding of the vitamin D receptor but increase the affinity to vitamin D-binding protein. The effects of many C-11-substituted 1 alpha,25-(OH)2D3 analogs on HL-60 cell differentiation exceeded their activity on calcium metabolism.

Plasma 1,25 dihydroxycholecalciferol and its free index are potentiated by ovulation dependent factors and shell formation induced hypocalcemia in the laying hens.

The time-course of the changes in blood ionized calcium, and in plasma 1,25 dihydroxycholecalciferol (1,25(OH)2D3) concentrations and its free index were studied in hens following suppression and resumption of shell formation and throughout the laying cycle in hens laying hard-shelled eggs, in hens fed a low or normal calcium diet and in hens laying shell-less eggs. The respective roles of the calcium needs for shell formation and of the reproductive status in regulation of 1,25(OH)2D3 production were analysed. Plasma 1,25(OH)2D3 decreased 3 hr after suppression of shell formation following premature egg expulsion and remained lower than that of hens laying hard-shelled eggs when premature expulsion of the eggs was continued for several days. Circulating 1,25(OH)2D3 tended to increase progressively when shell formation was resumed. Ablation of the parathyroid glands abolished this increase. In hens laying hard-shelled eggs, the plasma 1,25(OH)2D3 was higher during the period of shell secretion. Feeding hens a low calcium diet (1.2%) caused a marked increase in the plasma 1,25(OH)2D3. Ionized calcium levels tended to show reciprocal changes to plasma 1,25(OH)2D3 decreasing when calcification took place and increasing after its suppression. In hypercalcemic hens laying shell-less eggs and fed a 3.5% Ca diet, the plasma 1,25(OH)2D3 was at a high level 4 hr after ovulation and diminished thereafter. This additive stimulation does not, therefore, involve the parathyroid gland and may involve hormonal changes induced by ovulation. Vitamin D binding protein (DBP) in the plasma was at a high level in mature hens and was not affected by shell formation. Consequently, the free 1,25(OH)2D3 index fluctuated in parallel with total level of this hormone in mature hens. It is concluded that the calcium demand for shell formation modulates, in the short term, plasma 1,25(OH)2D3, via the homeostatic regulation of blood calcium by PTH, but that a large part of its increase is independent of PTH and is associated with the endocrine events concomitant with ovulation.

Vitamin D analogs with low affinity for the vitamin D binding protein: enhanced in vitro and decreased in vivo activity.

The affinity of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] and analogs with side-chain modifications [MC 903 or calcipotriol, MC 1147 or 24,24-dihomo-1 alpha,25-(OH)2D3 and 1,25-(OH)2-16ene-23yne-D3] for the vitamin D receptor and the serum vitamin D binding protein (DBP) were compared. The affinity of MC 903 for the receptor from chick and rat duodenum or from human peripheral blood mononuclear cells or HL-60 cells varied between 60 and 100% relative to the affinity of 1,25-(OH)2D3. The relative affinity of 1,25-(OH)2-16ene-23yne-D3 and MC 1147 varied for the same receptors between 45-70 and 3.5-25%, respectively. The relative affinity of MC 903 for human DBP was 30-fold decreased, whereas the two other analogs did not bind to DBP at all even in more than 1000-fold excess. The in vitro biologic activity of 1 alpha,25-(OH)2D3 on phytohemagglutinin-stimulated normal human lymphocyte proliferation was markedly inhibited by the addition of physiologic amounts of DBP to the cell culture medium. No such inhibition was observed when MC 903 or 1147 was evaluated similarly. DBP therefore reversed the rank order of the in vitro potency of these analogs. Intramuscular injections for 10 consecutive days to vitamin D-deficient chicks demonstrated a greater than or equal to 100-fold lower biologic activity of MC 903, MC 1147, and 1,25-(OH)2-16ene-23yne-D3 compared to that of 1 alpha,25-(OH)2D3 as evaluated by serum calcium and osteocalcin concentrations, as well as by duodenal calbindin D28K and bone calcium content.(ABSTRACT TRUNCATED AT 250 WORDS)

Photoaffinity labeling of human serum vitamin D binding protein and chemical cleavages of the labeled protein: identification of an 11.5-kDa peptide containing the putative 25-hydroxyvitamin D3 binding site.

In this paper, we describe photoaffinity labeling and related studies of human serum vitamin D binding protein (hDBP) with 25-hydroxyvitamin D3 3 beta-3'-[N-(4-azido-2-nitrophenyl)amino]propyl ether (25-ANE) and its radiolabeled counterpart, i.e., 25-hydroxyvitamin D3 3 beta-3'-[N-(4-azido-2-nitro-[3,5-3H]phenyl)amino]propyl ether (3H-25-ANE) (Ray et al., 1986, 1991). We have carried out studies to demonstrate that (1) 25-ANE competes with 25-OH-D3 for the binding site of the latter in hDBP and (2) 3H-25-ANE is capable of covalently labeling the hDBP molecule when exposed to UV light. Treatment of a sample of purified hDBP, labeled with 3H-25-ANE, with BNPS-skatole produced two Coomassie Blue stained peptide fragments, and the majority of the radioactivity was associated with the smaller of the two peptide fragments (16.5 kDa). On the other hand, cleavage of the labeled protein with cyanogen bromide produced a peptide (11.5 kDa) containing most of the covalently attached radioactivity. Considering the primary amino acid structure of hDBP, this peptide fragment (11.5 kDa) represents the N-terminus through residue 108 of the intact protein. Thus, our results tentatively identify this segment of the protein containing the binding pocket for 25-OH-D3.

Synthesis of 25-hydroxyvitamin D3 3 beta-3'-[N-(4-azido-2-nitrophenyl)amino]propyl ether, a second-generation photoaffinity analogue of 25-hydroxyvitamin D3: photoaffinity labeling of rat serum vitamin D binding protein.

Vulnerability of 25-hydroxy-[26,27-3H]vitamin D3 3 beta-N-(4-azido-2-nitrophenyl)glycinate, a photoaffinity analogue of 25-hydroxyvitamin D3 (25-OH-D3) (Ray et al., 1986) toward standard conditions of carboxymethylation promoted us to synthesize 25-hydroxyvitamin D3 3 beta-3'-[N-(4-azido-2-nitrophenyl)amino]propyl ether (25-ANE), a hydrolytically stable photoaffinity analogue of 25-OH-D3, and 25-hydroxyvitamin D3 3 beta-3'-[N-(4-azido-2-nitro-[3,5-3H]phenyl)amino] propyl ether (3H-25-ANE), the radiolabeled counterpart of 25-ANE. Competitive binding assays of 25-OH-D3 and 25-ANE with rat serum demonstrated that 25-ANE competes for the 25-OH-D3 binding site in rat serum vitamin D binding protein (rDBP). On the other hand, UV exposure of a sample of purified rat DBP (rDBP), preincubated in the dark with 3H-25-ANE, covalently labeled the protein. However, very little covalent labeling was observed in the absence of UV light or in the presence of a large excess of 25-OH-D3. These results provide strong evidence for the covalent labeling of the 25-OH-D3 binding site in rDBP by 3H-25-ANE.

Distribution of group-specific component/vitamin-D-binding protein subtypes in Saudi Arabia.

The distribution of group-specific component (Gc) phenotypes and the Gc allele frequencies were investigated in 1,082 individuals from five different provinces of Saudi Arabia by the combined use of isoelectric focusing and immunofixation. Between provinces variations in the Gc allele frequencies were found. Gc1S decreased and Gc1F increased from the northwest to the southeast in Saudi Arabia. The overall frequencies in Saudi Arabia were 0.236 for Gc1F, 0.610 for Gc1S, 0.150 for Gc2 and 0.004 for rare alleles. The observed frequencies did not differ significantly from those found in other population samples from the Middle East. In nine samples rare Gc variants were found.